Although considerable information about neuronal circuits has been generated from experiments where retrograde transsynaptic tracing has been performed using Rabies Virus, there is no comparable technique for transsynaptic tracing in the anterograde direction. The purpose of this grant is to optimize and validate a method for mediating anterograde transsynaptic tracing from genetically or physiologically determined cells. This method is monosynaptic, only marks cells that are postsynaptic to the starter cells and has negligible toxicity. To validate this method we will use a combination of immunocytochemistry, optogenetics and electrophysiology, and we will perform experiments both in vitro and in vivo. In addition, we will develop methods for tracing both excitatory and inhibitory transsynaptic connections. This method has the following advantages over current methods of transsynaptic tracing: 1. All steps of the process are well-defined and thus can be independently tested and optimized. 2. It is mediated by a single receptor, so that when applied to neurons such as dopaminergic neurons that release more than one neurotransmitter, it should be possible to independently trace pathways mediated by each transmitter. 3. It should be generalizable to any postsynaptic receptor, allowing many different pathways to be explored. 4. The receptor that mediates transsynaptic labeling is physiologically relevant ? only electrically active synapses, and not silent synapses, will mediate postsynaptic labeling.